title: Rationalized DNA sequencing-based protocol for genotyping patients receiving coumarin therapy.
abstract: During the last decade genetic factors affecting coumarin therapy have been extensively investigated. The most important genes appear to be CYP2C9 and VKORC1, and different studies have shown that DNA testing can dramatically improve the safety and effectiveness of the therapy. However, the implementation of pharmacogenetic testing in everyday practice is still not a reality. Facilities and ability to get results before the start of therapy are very important. The implementation of specific methodology and equipment for particular type of diagnostics can represent a serious, even impossible, financial hurdle to overcome (especially in developing countries). For this reason, the use of every tool that contributes to rationalization of the existing methods can be a considerable asset. Therefore, we set the goal to rationalize our current DNA sequencing based protocol for analysis of the VKORC1 c.-1639G> A, CYP2C9*2 and CYP2C9*3 variant alleles, in order to obtain shorter and easier procedure. Simplification of the protocol was achieved by setting up multiplex PCR and omitting DNA extraction. This rationalization of the existing DNA sequencing based procedure allows getting results in 12 hours. The new protocol was tested on 118 samples. Obtained results have shown full accordance to those obtained with previous, non-modified protocol. Therefore, given the circumstances, we consider that protocol for pharmocogenetic testing should be made more accessible - both to doctors and patients. It is one of the prerequisites in order to make genotyping prior to the therapy common practice.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Serbia.
authors.forename: Ljiljana B
authors.initials: LB
authors.lastname: Rakicevic
authors.forename: Jelena S
authors.initials: JS
authors.lastname: Kusic-Tisma
authors.forename: Mirjana K
authors.initials: MK
authors.lastname: Kovac
authors.forename: Dragana T
authors.initials: DT
authors.lastname: Backovic
authors.forename: Dragica P
authors.initials: DP
authors.lastname: Radojkovic
mesh_terms: D000925:Anticoagulants / Q000627:therapeutic use*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D003374:Coumarins / Q000627:therapeutic use*; D065729:Cytochrome P-450 CYP2C9; D005787:Gene Frequency; D060005:Genotyping Techniques; D006239:Haplotypes; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013923:Thromboembolism / Q000235:genetics / Q000517:prevention & control*; D064417:Vitamin K Epoxide Reductases / Q000235:genetics
chemical_list: D000925:Anticoagulants; D003374:Coumarins; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases